99mTc-Ethylenediaminediacetic acid/hydrazinonicotinic acid-[dGlu1, desGlu2–6]minigastrin (99mTc-EDDA/HYNIC-MG11) is a radiolabeled peptide developed for single-photon emission computed tomography (SPECT) imaging of tumors that express the gastrin/cholecystokinin-2 (CCK-2) receptor (1). 99mTc is a gamma emitter with a physical half-life (t½) of 6.01 h.
The gastrointestinal peptides gastrin and CCK have various regulatory functions in the brain and gastrointestinal tract (2). Gastrin and CCK have the same COOH-terminal pentapeptide amide sequence, which is the biologically active site (3). Human gastrin is a peptide composed of 34 amino acids and also exists in several C-terminal truncated forms (4). These C-terminal truncated forms include minigastrin, which is a 13-residue peptide with the sequence of LEEEEEAYGWMDF-NH2. CCKs exist in a variety of biologically active molecular forms that are derived from a precursor molecule comprising 115 amino acids (5). These forms range from 4 to 58 amino acids in length and include sulphated and unsulphated CCK-8, which has the structure DYMGWMDF-NH2. They bind to and act through transmembrane G-protein–coupled receptors (6). Two different CCK receptor subtypes have been identified in normal tissues. CCK-1 (CCK-A, alimentary) receptors have low affinity for gastrin, and CCK-2 (CCK-B, brain) receptors have high affinity for gastrin (5). They also differ in terms of molecular structure, distribution, and affinity for CCK. These receptors have also been found to be expressed or overexpressed on a multitude of tumor types (6). CCK-2 receptors have been found most frequently in medullary thyroid carcinoma, small-cell lung cancers, astrocytomas, and stromal ovarian cancers (2). CCK-1 receptors have been identified in gastroenteropancreatic tumors, meningiomas, and neuroblastoma.
Reubi et al. (7) designed a series of radiolabeled CCK-8 peptides that showed high specificity for potential in vivo imaging of tumors expressing CCK-2 receptors. Because of its favorable physical properties, 99mTc is still the radionuclide of choice for routine clinical applications (8). Hydrazinonicotinic acid (HYNIC) is a bifunctional coupling agent for 99mTc labeling of peptides that can achieve high specific activities without interfering with the amino acid sequence responsible for receptor binding (9-11). In this approach, 99mTc is bound to the hydrazine group, and other coordination sites are occupied by one or more coligands. The choice of coligand can influence the stability and hydrophilicity of the radiolabeled peptide. Using the HYNIC labeling strategy and ethylenediaminediacetic acid (EDDA) or tris(hydroxymethyl)-methylglycine (tricine) as the coligand, von Guggenberg et al. (12) reported the success of radiolabeling the MG0 peptide (d-Glu-Glu5-Ala-Tyr-Gly-Trp-Met-Asp-PheNH2). The 99mTc-EDDA/HYNIC-MG0 peptide showed high tumor uptake in nude mice bearing AR42J tumors, but it also has very high kidney activity. To minimize kidney uptake, the same research group designed a shortened peptide analog by depletion of the five glutamic acid molecules in positions 2–6 to produce 99mTc-EDDA/HYNIC-[dGlu1, desGlu2–6]minigastrin (99mTc-EDDA/HYNIC-MG11) (1).
von Guggenberg et al. (1) reported the radiosynthesis of 99mTc-EDDA/HYNIC-MG11 by both the EDDA direct labeling and tricine/EDDA exchange labeling methods. Tricine, EDDA, and HYNIC-MG were obtained commercially. The purity of HYNIC-MG was >95%. In the direct radiolabeling procedure, HYNIC-MG (10 μg) was mixed and incubated with EDDA, 400 MBq (10.8 mCi) 99mTc pertechnetate (99mTcO4−), and stannous chloride in a total volume of 1 ml at pH 6 and 80ºC for 30 min. High-performance liquid chromatography (HPLC) analysis showed a radiochemical purity of >90%. The specific activity was >70 GBq/μmol (>1.89 Ci/μmol). In the tricine/EDDA exchange labeling method, HYNIC-MG was incubated with EDDA/tricine solution (20 mg/ml tricine, 10 mg/ml EDDA), 99mTcO4−, and stannous chloride (1 mg/ml) in a total volume of 2 ml at pH 6 and 100ºC for 10 min. The labeling yield was <90% and the radiochemical purity was <90%. HPLC analysis showed that only incomplete conversion of the 99mTc-tricine complex into the 99mTc-EDDA complex was achieved. Further purification was achieved by solid-phase extraction with the use of a C18-Sep-Pak-Light cartridge. The authors suggested that the 99mTc-EDDA-HYNIC-MG11 was composed of two EDDA coligand molecules per peptide molecule. They also suggested that impurities exposed by HPLC analysis were products of isomerism and oxidative side products.
In Vitro Studies: Testing in Cells and Tissues
The in vitro stability of 99mTc-EDDA/HYNIC-MG11 purified by solid-phase extraction was tested by incubating the radiolabeled peptide (200–1,000 pmol/ml) in phosphate-buffered saline (PBS, pH 7.4), PBS that contained 10,000-fold molar excess of cysteine (cysteine PBS), or fresh human plasma at 37ºC for up to 24 h (1). At 4 h, HPLC analysis showed that the amounts of intact 99mTc-EDDA/HYNIC-MG11 were 98.6%, 91.2%, and 98.1% in the PBS, cysteine PBS, and plasma, respectively. At 24 h, the amounts of intact radiolabeled peptide were 95%, 85.8%, and 89.4% in the PBS, cysteine PBS, and plasma, respectively. Invitro metabolic stability studies of 99mTc-EDDA/HYNIC-MG11 were conducted in rat kidney and liver homogenates (1). The studies showed a very rapid degradation of 99mTc-EDDA/HYNIC-MG11. After 30 min of incubation in the liver homogenates, <5% intact peptide remained. Incubation in the kidney homogenates showed that the degradation was more rapid with <5% intact peptide remaining at 10 min.
The binding affinities of unlabeled EDDA/HYNIC-MG11 and 99mTc-EDDA/HYNIC-MG11 were tested in competition assays against 125I-Tyr12gastrin by using rat pancreatic tumor AR42J cell membranes as the source of gastrin receptors (1). The inhibition constant (IC50) of the unlabeled HYNIC-MG11 was <2 nM, and the dissociation constant (Kd) of 99mTc-EDDA/HYNIC-MG11 was determined to be 3.97 nM. AR42J cell internalization studies (n = 3) showed that 12 ± 0.21% of the total radioactivity was internalized after 120 min of incubation (1). The radioligand was rapidly internalized after binding with >80%, and 94.4 ± 1.22% internalized fractions of the bound activity were achieved after 15 min and 120 min of incubation, respectively. Externalization experiments (efflux studies; n = 3) showed a very small amount of released radioactivity: 4.02 ± 0.21% after 1.5 h of incubation.
von Guggenberg et al. (1) conducted biodistribution studies of 99mTc-EDDA/HYNIC-MG11 in nude mice bearing AR42J tumors. Each mouse received 1 MBq (27 μCi) 99mTc-EDDA/HYNIC-MG11 (~0.05 μg) radioactivity. In general, the radiolabeled peptide was mainly excreted by the kidneys and was rapidly eliminated from most organs. The radioactivity levels for the tumor were 4.77 ± 0.72% injected dose/g (% ID/g) and 7.11 ± 0.22% ID/g at 1 h and 4 h, respectively. These levels decreased to 1.37 ± 0.54% ID/g and 1.10 ± 0.26% ID/g when 50 μg human minigastrin I (MGh) was coinjected with the radioligand. The kidney activities were 2.44 ± 0.97% ID/g and 1.96 ± 0.14% ID/g at 1 h and 4 h, respectively. These levels slightly decreased to 2.18 ± 0.40% ID/g and 1.54 ± 0.16% ID/g with the blocking of MGh. This was a 98% reduction in the kidney radioactivity level when compared with that of the 99mTc-EDDA/HYNIC-MG0 peptide. The other major organ radioactivity levels (% ID/g) at 4 h were 1.26 ± 0.07 (liver), 0.90 ± 0.23 (intestine), 0.46 ± 0.23 (stomach), 0.49 ± 0.10 (spleen), 0.15 ± 0.05 (lung), 0.11 ± 0.14 (blood), 0.09 ± 0.04 (pancreas), and 0.02 ± 0.02 (muscle). A significant reduction in radioactivity levels as a result of MGh coinjection was observed in receptor-expressing organs (stomach and pancreas).
Other Non-Primate Mammals
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- Reubi J.C., Schaer J.C., Waser B. Cholecystokinin(CCK)-A and CCK-B/gastrin receptors in human tumors. Cancer Res. 1997;57(7):1377–86. [PubMed: 9102227]
- Aly A., Shulkes A., Baldwin G.S. Gastrins, cholecystokinins and gastrointestinal cancer. Biochim Biophys Acta. 2004;1704(1):1–10. [PubMed: 15238241]
- Mather S.J., McKenzie A.J., Sosabowski J.K., Morris T.M., Ellison D., Watson S.A. Selection of radiolabeled gastrin analogs for Peptide receptor-targeted radionuclide therapy. J Nucl Med. 2007;48(4):615–22. [PMC free article: PMC2246928] [PubMed: 17401100]
- Wang H., Wong P.T., Spiess J., Zhu Y.Z. Cholecystokinin-2 (CCK2) receptor-mediated anxiety-like behaviors in rats. Neurosci Biobehav Rev. 2005;29(8):1361–73. [PubMed: 16120463]
- Behr T.M., Behe M.P. Cholecystokinin-B/Gastrin receptor-targeting peptides for staging and therapy of medullary thyroid cancer and other cholecystokinin-B receptor-expressing malignancies. Semin Nucl Med. 2002;32(2):97–109. [PubMed: 11965605]
- Reubi J.C., Waser B. Unexpected high incidence of cholecystokinin-B/gastrin receptors in human medullary thyroid carcinomas. Int J Cancer. 1996;67(5):644–7. [PubMed: 8782652]
- Aloj L., Panico M.R., Caraco C., Zannetti A., Del Vecchio S., Di Nuzzo C., Arra C., Morelli G., Tesauro D., De Luca S., Pedone C., Salvatore M. Radiolabeling approaches for cholecystokinin B receptor imaging. Biopolymers. 2002;66(6):370–80. [PubMed: 12658724]
- Liu S., Edwards D.S. 99mTc-Labeled Small Peptides as Diagnostic Radiopharmaceuticals. Chem Rev. 1999;99(9):2235–68. [PubMed: 11749481]
- Decristoforo C., Melendez-Alafort L., Sosabowski J.K., Mather S.J. 99mTc-HYNIC-[Tyr3]-octreotide for imaging somatostatin-receptor-positive tumors: preclinical evaluation and comparison with 111In-octreotide. J Nucl Med. 2000;41(6):1114–9. [PubMed: 10855644]
- Barrett J.A., Crocker A.C., Damphousse D.J., Heminway S.J., Liu S., Edwards D.S., Lazewatsky J.L., Kagan M., Mazaika T.J., Carroll T.R. Biological evaluation of thrombus imaging agents utilizing water soluble phosphines and tricine as coligands when used to label a hydrazinonicotinamide-modified cyclic glycoprotein IIb/IIIa receptor antagonist with 99mTc. Bioconjug Chem. 1997;8(2):155–60. [PubMed: 9095355]
- von Guggenberg E., Behe M., Behr T.M., Saurer M., Seppi T., Decristoforo C. 99mTc-labeling and in vitro and in vivo evaluation of HYNIC- and (Nalpha-His)acetic acid-modified [D-Glu1]-minigastrin. Bioconjug Chem. 2004;15(4):864–71. [PubMed: 15264875]
Created: May 4, 2007; Last Update: June 7, 2007.
National Center for Biotechnology Information (US), Bethesda (MD)
The MICAD Research Team. 99mTc-Ethylenediaminediacetic acid/hydrazinonicotinic acid-[dGlu1, desGlu2–6]minigastrin. 2007 May 4 [Updated 2007 Jun 7]. In: Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.